This invention relates generally to ligands for CD7 and uses thereof to treat diseases, isolated polynucleotide molecules encoding such ligands, purified ligands, and methods of screening for molecules that affect the interaction between CD7 and its cognate ligand K12.
Cellular change is often triggered by the binding of an extrinsic element, such as a ligand, to the extracellular domain of a receptor protein. This binding can result in cellular changes by activating and controlling intracellular signaling cascades and cell regulatory mechanisms. As such, understanding the binding interaction between the ligand and its receptor protein can be of great interest to the scientific community. A greater understanding of the interaction would enable one skilled in the art to modulate the resulting signaling cascade governed by the ligand/receptor interaction by selecting agents for co-stimulation or inhibition of the binding of the ligand to its receptor. Furthermore, in the instances of bi-directional or reverse signaling, the interaction would not only activate the signaling cascade of the receptor but would also trigger a signaling cascade in a cell bearing the ligand (Wiley et al., J. of Immun., 3235-39 (1996)). Thus, understanding the interaction between a receptor and ligand can lead to therapeutic treatments involving the inhibition or enhancement of either one or both of the receptor activity or ligand activity.
CD7 is a 40 kDa protein found primarily on hematopoietic cells. CD7 is expressed on mature T and natural killer (NK cells), as well as on progenitors of T, B, NK, and myeloid cells (Aruffo, Embo J. 6:3313 (1987); Yoshikawa, Immunogenetics 33:352 (1991); Yoshikawa, Immunogenetics 37:114 (1993); Yoshikawa, Immunogenetics 41:159 (1995)) and on intestinal intraepithelial lymphocytes (Spencer, Gut 30:339 (1989); Eiras, Cytometry 34:95 (1998)). In addition, CD7 is thought to be a marker for one of the earliest stages of T cell development (reviewed in Barcena, Leuk Lymphoma 17:1 (1995) and Haynes, Semin Immunol 2:67 (1990)).
Although all of the functions of the CD7 protein in the immune system are unknown, signaling through CD7 has been reported using anti-CD7 antibodies (Carrera, J.Immunol 141:1919 (1988); Rabinowich, J.Immunol Vol. 153:3504 (1994)), indicating that the cytoplasmic domain of CD7 must contain some signal tranducing elements or that it complexes with a protein containing such elements. Recombinant soluble CD7 also inhibits antigenic- and alloantigenic-induced T cell proliferation (Leta, Cell Immunol 165:101 (1995)).
Previous efforts to identify a cognate for CD7 have not been successful, although a putative ligand has reportedly been detected in serum (Leta, Cell Immunol 173:15 (1996)). The extracellular domain of CD7, expressed by either mammalian or insect cells, has been shown to interact with some specificity with Con A (Id.). This interaction is at least partly mediated through carbohydrate residues on CD7 since treatment of the extracellular domain of CD7 with glycosidases specifically reduces binding of Con A to CD7.
A recent study used model systems to investigate whether CD7 deficient mice are resistant to LPS-induced shock. According to the study, CD7 deficient mice were totally resistant to low-dose LPS-induced shock syndrome and were partially resistant to high-dose LPS-induced shock syndrome. Thus, this data indicates that CD7 is involved in the LPS-induced shock pathway (Sempowski et al., J. Exp. Med. 189, 1011-1016 (1999)).
Given the role CD7 plays in signal transduction (including its mediation of the LPS-induced shock pathway), there is a need in the art for the identification and understanding of the interaction of CD7 with its cognate ligand or binding partner. Further, there is a need for the development of assays and therapeutic methods using the interaction between CD7 and its binding ligand.
The K12 gene (also known as SECTM1) was originally identified (Slentz-Kesler, Genomics 47:327(1998)) as being directly 5xe2x80x2 of the locus encoding the human CD7 gene on human chromosome 17 (Osada, Cytogenet Cell Genet (1988)). The 3xe2x80x2 end of the K12 gene is about 5 kb upstream of the start of the human CD7 gene, and both genes are transcribed in the same direction (Slentz-Kesler, 1998). The K12 gene encodes a transmembrane protein with two short regions in the extracellular domain with weak similarity to Ig-like domains. Both membrane-bound (localized to the Golgi apparatus) and secreted forms of the protein were observed (Id.). In humans, the protein is primarily expressed in spleen, prostate, testis, small intestine, and in peripheral blood leukocytes (Id). However, no known function for the K12 protein has yet been identified.
The invention is based, in part, on the discovery that the cognate for the K12 protein is CD7. Thus, K12 is the previously unknown ligand or binding partner for CD7. Accordingly, an aspect of the invention is the use of the K12 protein, and polynucleotides encoding the same, in methods of modulating CD7 and K12 signaling events, and in treating diseases. The invention also provides isolated polynucleotide molecules encoding novel K12 proteins, and the recombinant and/or purified K12 polypeptides encoded by such polynucleotides.
Other aspects of the invention include methods of screening for compounds, which are referred to as xe2x80x9ctest compoundsxe2x80x9d or xe2x80x9ccandidate moleculesxe2x80x9d that affect the K12/CD7 interaction. In one aspect, the method comprises the steps of forming a composition comprising a CD7 protein, a K12 protein, and the test compound; assaying for the level of interaction of the CD7 protein and the K12 protein; and comparing the level obtained in the presence of the test compound to that obtained in the absence of the test compound, such that if the level obtained differs, a compound that affects the interaction of the CD7 protein and the K12 protein is identified. At least one of the CD7 protein and the K12 protein can be labeled with a detectable moiety. Preferred test compounds are small organic molecules, antibodies, and small peptides. One of the CD7 protein or the K12 protein can be soluble, and the other can be bound, although alternative assay formats are possible and well known. The test compound can be added to the composition after addition of the CD7 protein and the K12 protein, before both proteins are added, or after one protein is added and before the other is added.
In another aspect, the screening methods of the invention comprise forming a composition comprising the test compound, the K12 protein and cells expressing CD7; determining the level of biological activity of the K12 protein in the composition; and comparing the level of biological activity with that which occurs in the absence of test compound, wherein a difference in the level of biological activity indicates that the test compound affects the biological activity of a K12 protein. Biological activity of K12 can be assayed in any number of ways, for example, by determining the phosphorylation state of intracellular proteins, by determining the activation of NK cells and by determining the production of interferon gamma and/or GM-CSF. In a related aspect, the cells express K12 and soluble CD7, or cells expressing CD7, is used.
The present invention also provides a screening method for identifying candidate molecules that enhance or inhibit the interaction between CD7 and K12, or that prevent or inhibit dissociation of a complex formed by CD7 and K12. This screening method involves contacting a mixture of cells which express CD7 and cells which express K12 with a candidate molecule, measuring cellular responses, and detecting the ability of the candidate molecule to inhibit or enhance the interaction between CD7 and K12 or inhibit the dissociation of the complex formed by CD7 and K12. Successful inhibition indicates that the candidate molecule is an antagonist. Increased activation of CD7 or K12 indicates that the candidate molecule is an agonist.
In another aspect, the invention provides antagonists and agonists of the interaction between CD7 and K12. In yet a further aspect, the invention provides for a therapeutic use of agonists and antagonists of the interaction between CD7 and K12 in the treatment of diseases modulated by CD7 and/or K12. In still a further aspect, the invention provides for a therapeutic use of K12 in the treatment of disease modulated by CD7. In yet still a further aspect, the invention provides for a therapeutic use of CD7 in the treatment of disease modulated by K12.
SEQ ID NO:1 is the cDNA sequence of human CD7.
SEQ ID NO:2 is the amino acid sequence of human CD7.
SEQ ID NO:3 is the nucleic acid sequence of human K12.
SEQ ID NO:4 is the amino acid sequence of human K12.
SEQ ID NO:5 is the cDNA sequence of murine CD7.
SEQ ID NO:6 is the amino acid sequence of murine CD7.
SEQ ID NO:7 is the nucleic acid sequence of murine K12.
SEQ ID NO:8 is the amino acid sequence of murine K12.